Clostridium difficile is an emerging pathogen that causes severe and sometimes deadly colitis. Epidemiological evidence indicates that both the severity and incidence of C. difficile-associated disease (CDAD) are increasing at an alarming rate. Available treatment is inadequate and there are no licensed vaccines. Type IV pili (T4Ps), surface appendages that play important roles in infections caused by diverse bacterial pathogens, have been exploited for vaccine development. Recently, T4Ps were discovered in C. perfringens and the genes for T4Ps are present in C. difficile. Our overall hypothesis states that an immune response against the major pilin will protect against colonization and CDAD. To test this hypothesis we will first determine which pilin gene encodes the major structural subunit of the C. difficile T4P and then investigate the efficacy of immunization with purified pilin in prevention of C. difficile colonization and disease in a murine model. The proposed research is highly innovative because it will examine the potential of a novel antigen usually identified with Gram-negative bacteria to protect against colonization by a Gram-positive anaerobe. A result indicating a protective effect will have a profound effect on research and strategies for prevention and therapy for clostridial infections. The investigators have an established record of productivity studying T4P and using animal models of disease and the environment at the University of Maryland Baltimore is ideal for the proposed research. Given the indispensible role of T4Ps for other infections, the immunogenicity of pilin proteins, and the success of other T4P vaccines, we expect that a C. difficile pilin vaccine (alone or in combination with a toxoid vaccine) will generate a potent protective immune response against this potentially lethal infection. Demonstration of protection in a highly relevant animal model will pave the road to further preclinical and clinical testing.